干扰肿瘤微环境对细胞毒性T淋巴细胞瘤体内归巢和杀伤活性的影响

Effect of Interfering Tumor Microenvironment on Homing and Killing Activity of Cytotoxic T Lymphocytes

目的探讨应用低剂量环磷酰胺(cyclophosphamide,CYC)干扰荷瘤小鼠体内微环境对改善过继回输的细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)瘤体内归巢和杀伤活性的作用。方法建立C57BL/6荷瘤小鼠模型并分为对照组、CYC注入(CYCI)组、0.9%Na Cl溶液注入(NSI)+CTL组和CYCI+CTL组,每组40只,分别给CYCI组、CYCI+CTL组每只小鼠腹腔注入CYC(20mg/kg),NSI+CTL组小鼠注入等量0.9%Na Cl溶液。检测注射前后不同时间小鼠瘤体内调节性T细胞(Treg)、白细胞介素-10(IL-10)和转化生长因子-β(TGF-β)水平变化。于注射后第4天回输CFSE-CTL,每只0.2 ml,含5×l06个细胞。流式检测对比NSI+CTL组和CYCI+CTL组瘤体内CFSE-CTL比例。结果 NSI+CTL组小鼠Treg、IL-10、TGF-β水平随着瘤体增长而逐渐增高;CYCI+CTL组比NSI+CTL组回输的CFSE-CTL在肿瘤内归巢明显增加和持久(P<0.05)。各组肿瘤体积除对照组和CYCI组外,其余各组间差异均有统计学意义(P<0.05)。结论提前应用低剂量CYC干预荷瘤小鼠体内微环境,可降低瘤体内Treg、IL-10和TGF-β水平,使过继回输的CTL在肿瘤中的归巢聚集明显增强,杀伤肿瘤效率提高。

Objective To explore the effect of interfering tumor microenvironment with low-dose cyclophosphamide (CYC) administration on homing and killing activity of adoptive transfused cytotoxic T lymphocytes in adoptive immunotherapy.Methods Melanoma-bearing mice models were established and randomly divided into four groups:control group,CYC injection group (CYCI),the solution of 0.9％ w/v of NaC1 injection plus CTL infusion group (NSI+CTL) and CYC injection plus CTL infusion group (CYCI+CTL),40 cases in each group.CYC (20 mg/kg) were intraperitoneally injected in mice of CYCI group and CYCI+CTL group.The same amount of the solution of 0.9％ w/v of NaC1 was injected in mice of NSI+CTL group.The levels of Treg,TGF-β and IL-10 were detected at the different time points before and after CYC or the solution of 0.9％ w/v of NaC1 injection.The cultured CFSE-CTLs were transfused at the 4th day after injection,5×106 cells per mouse.Then,CFSE-CTLs ratio was analyzed by FCM.Results In NSI+CTL group,the levels of Treg,TGF-β and IL-10 were all increased continuously with tumor growth.CFSE-CTLs ratio in CYCI+CTL group was significantly higher and lasted longer than that in NSI+CTL group (P＜0.05).There was significant difference among every two group,except control group and CYCI group(P＜0.05).Conclusion Tumor microenvironment in tumor-bearing mice could be effectively intervened by low-dose CYC administration in advance,with decreased Tregs,IL-10,and TGF-β levels.As a result,more transferred CTLs homing leads to the strenthened killing efficacy.